SUMMARY' Interruption of antiretroviral therapy (ART) during HIV infection leads to a very quick virus rebound even in individuals that were aviremic for years. Developing strategies that would eliminate the need of life-long therapy is the current major objective of the biomedical community engaged in research on HIV infection and AIDS. To achieve this goal, clearance of viral reservoirs that persist despite antiviral immune responses and ART is the critical step. Infection of tissues macrophages occurs in many organs and some reports support the contribution of macrophages to viral reservoirs during ART. As macrophages appear more difficult to eliminate by virus specific CD8+ T cells, it is important to establish whether infected macrophages are indeed part of the pool of infected cells that contribute to virus rebound when ART is interrupted. Toward this goal, we propose to evaluate the frequency of SIV+ myeloid cells in lungs, bone marrow, spleen, lymph nodes, intestinal mucosa and brain after 54-59 weeks of suppressive ART in SIV-infected rhesus macaques and estimate SIV sequence evolution in macrophages compared to the evolution observed in T cells and to the sequence diversity of virus inoculum and of initial plasma viremia. The novel cell associated SIV RNA and DNA single genome sequencing (CARD-SGS) assay will be employed In T-cell and macrophage purified populations obtained from the tissues of suppressed and viremic animals mentioned above. We also propose to evaluate the levels SIV protein expression in myeloid reservoirs and the infectivity of virus present in tissue SIV+ macrophages during ART. For these experiments the combination of RNAScope/DNAScope with confocal microscopy will be utilized in tissue sections. Replication-competent SIV will also by evaluated in tissue-derived purified cell populations, using the quantitative viral outgrow assay (QVOA). Obtaining a complete picture of virus reservoirs appears imperative to better understand the basis of chronic viral diseases and towards the goal of a HIV cure. !